Flower color is an important character when people appreciate or buy flowers and ornamental plants, and the flowers with various colors have been bred traditionally. It is rather rare that a single kind of species has flowers with all colors, and this is due to the fact that biosynthesis of pigments (flower pigments) expressed as the flower colors is genetically defined. It has been substantially impossible to make the flowers with all colors in the target species by crossing because gene resources available for cross breeding are limited to related interfertile species. Only recently, it has become possible to modify the flower color by taking advantage of the gene recombination technology, obtaining a gene of an enzyme involved in synthesis of the flower pigment, and expressing the gene in other species (e.g., Non-patent Document 1, Non-patent Document 2).
Among the flower colors, an orange, red, purple and blue colors are primarily derived from flavonoid referred to as anthocyanin. A yellow color is often derived from compounds such as carotenoid and betalain other than flavonoid, but the yellow color of some plant species is derived from flavonoid. For example, it is known that a glycoside having sugar at position 2′ of 4,2′,4′,6′-tetrahydroxychalcone (THC) is present in petals of a yellow carnation (e.g., Non-patent Document 3). As chalcones, glycosides of THC, butein, and isoliquitigenin are known. As aglycones of the glycosides, The THC is contained in carnation, morning glory, peony, aster and strawflower, 3,4,2′, 4′, 6′-pentahydroxychalcone is contained in snapdragon and statice, the butein is contained in cosmos and Jerusalem artichoke, and the butein and isoliquitigenin are contained in dahlia. In limited species such as snapdragon, yellow flower pigment referred to as aurones such as aureusidin and brackteatine is present. An absorption maximum of the aurones is 399 nm to 403 nm whereas the absorption maximum of the chalcones is 365 to 382 nm, and thus color tones of both are different (e.g., see Non-patent Document 4). Generally in plant cells, the chalcones and the aurones are stabilized by becoming sugar-transferred glycosides, migrate to and are accumulated in vacuoles. Biosynthetic pathways of anthocyanin are well studied, and enzymes involved in the biosynthesis of anthocyanin and genes encoding them are known (e.g., see Non-patent Document 5). Enzymes involved in the biosynthesis of the aurone and genes thereof have been reported (e.g., see Non-patent Document 6).
The biosynthetic pathways of the flavonoids are present widely in higher plants and common between the species. The THC is biosynthesized from three molecules of malonyl-CoA and one molecule of coumaroyl-CoA by catalysis of chalcone synthase. As shown in FIG. 1, The THC exhibits pale yellow color, but in the plant, it is rapidly converted to colorless naringenin by chalcone isomerase. The THC is also extremely unstable at pH around a neutral, and is converted to naringenin by spontaneously closing a ring. In order for THC to be present stably in the plant cell, i.e., stably exhibit the yellow color, it is described that it is necessary that glycosyltransfer takes place at position 2′ of THC and the THC is transported into the vacuole. Therefore, it has been believed that if a gene of the enzyme which transfers the sugar to the position 2′ of the THC can be obtained, flowers with yellow color can be made by expressing this enzyme gene in the plant and accumulating THC glycoside (e.g., see Non-patent Document 7).
However, it has been impossible to measure an activity of the enzyme which catalyzes a reaction to transfer the sugar, e.g., glucose to a hydroxyl group at the position 2′ of the chalcones including the THC. Conventionally, the activity of chalcone glycosyltransferase has been measured by labeling UDP-glucose with a radioisotope, performing an enzymatic reaction, subsequently extracting a produced glycoside with ethyl acetate and measuring a radioactivity in an extracted organic layer (e.g., see Non-patent Document 8). However, most glycoside of the THC is moved to an aqueous layer, and thus it is most likely that glucose whose radioactivity has been counted is unreacted free UDP-glucose which has been slightly eluted in the organic layer. Therefore, there has been a problem that the original activity of THC glycosyltransferase can not be measured accurately. Accordingly, the enzyme which catalyzes this glycosyltransfer reaction could not be purified, and thus the gene encoding the glycosyltransferase could not be cloned.
It is known that the petals also become the yellow color when a compound where hydroxyl group at position 2′ of THC is methylated is accumulated, but neither enzyme which catalyzes this methylation nor gene thereof are known. 6′-Deoxychalcone is contained in yellow varieties of the dahlia and cosmos. In legume, 6′-deoxychalcone is a precursor of 5-deoxyflavonoid and is biosynthesized by catalysis of chalcone synthase (CHS) and chalcone reductase (CHR). It has been reported that a CHR gene of alfalfa was introduced into petunia and then 6′-deoxychalcones such as butein were synthesized. However, when the CHR gene was introduced into the petunia with white flowers, extremely pale yellow color was observed in flower buds, but bloomed flowers were almost white. Thus, this attempt did not lead to production of industrially useful yellow flowers (e.g., see Non-patent Document 9).
Enzymes which catalyze the glycosyltransfer reaction of various flower pigment compounds including flavonoid to produce the glycosides are referred to as glycosyltransferases. The plants have the glycosyltransferases of various molecular species having specificity depending on kinds of the aglycone and the transferred sugar, and genes encoding them. Glucose transferase usually utilizes UDP-glucose as a glucose donor, and thus the glucose transferase includes a motif to bind to the UDP-glucose in an amino acid sequence thereof (e.g., Non-patent Document 10). It is known that there are 99 kinds of genes for the glycosyltransferases having this motif in Arabidopsis whose genomic structure has been already shown entirely (e.g., see Non-patent Document 11). The amino acid sequences and functions in some glycosyltransferases have been elucidated. Genes of an enzyme (UDP-glucose: flavonoid 3-glycosyltransferase) which catalyzes a reaction to transfer the glucose to the hydroxyl group at position 3 of the flavonoid or anthocyanidin have been obtained from maize, gentian and grape (e.g., see Non-patent Document 11). Genes of an enzyme (UDP-glucose: anthocyanin 5-glycosyltransferase) which catalyzes a reaction to transfer the glucose to the hydroxyl group at position 5 of anthocyanin have been obtained from perilla and verbena (e.g., see Non-patent Document 12).
From analyses of the amino acid sequences of these glucose transferases, it has been know that proteins having the same function which catalyzes the glucose transfer reaction are similar in amino acid sequences even when the plant species are different, i.e., the proteins form a family (e.g., see Non-patent Document 11). That is, it has been reported to obtain the enzymes (ortholog) having the same function as that of the glucose transferase where the amino acid sequence and catalysis of the glucose transfer reaction were demonstrated, from the other plant species. For example, the gene of UDP-glucose: anthocyanin 5-glycosyltransferase in the petunia was cloned using the gene of UDP-glucose: anthocyanin 5-glycosyltransferase in the perilla (e.g., see Non-patent Document 13). However, even at a current technical level, numerous trials and errors as well as difficulties are involved in acquisition of the gene of glycosyltransferase whose amino acid sequence or function is unknown. In particular, the flower of Arabidopsis is white, and no accumulation of chalcone glycoside having the sugar at position 2′ has been reported. Therefore, the gene can not be cloned by taking advantage of information for the glycosyltransferase genes of Arabidopsis whose genomic structure has been already determined entirely. Concerning the carnation, it has been reported that when mutation occurs in a chalcone isomerase gene and dihydroflavonol reductase gene, the THC glycoside is accumulated to exhibit the yellow color. In the cyclamen, it is also believed that the THC glycoside is accumulated by mutation of chalcone isomerase. Likewise, as the plants in which the chalcone glycoside having the sugar at position 2′ is accumulated, petunia pollen, Paeonia lactiflora, strawflower, China aster, cyclamen, evening primrose and periwinkle are known. It is also believed that the gene of the enzyme which transfers the sugar to the position 2′ of THC is expressed in numerous plants, particularly the plants which exhibit the yellow flower color.
Non-patent Document 1: Plant Cell Physiol., 39:1119 (1998)
Non-patent Document 2: Curr. Opin. Biotechnol., 12:155 (2001)
Non-patent Document 3: Phytochemistry 5:111 (1996)
Non-patent Document 4: Biohorti I 49-57, Seibundo Shinkosha (1990)
Non-patent Document 5: Comprehensive Natural Products Chemistry, Vol. I (ed., Sankawa) pages 713-748, Elsevier Amsterdam (1999)
Non-patent Document 6: Science 290:1163 (2000)
Non-patent Document 7: Biotechnology of Ornamental Plants (ed., Geneve, Preece and Merkle) pages 259-294, CAB International Wallingford, UK (1997)
Non-patent Document 8: Phytochemistry 17:53-56 (1978)
Non-patent Document 9: Plant J., 13:259 (1998)
Non-patent Document 10: Plant Physiol., 112:446 (2001)
Non-patent Document 11: J. Biol. Chem., 276:4338 (2001)
Non-patent Document 12: J. Biol. Chem., 274:7405 (1999)
Non-patent Document 13: Plant Mol. Biol., 48:401-11 (2002)